1. Field of the Invention
The present invention relates to an optical biosensor with a novel construction for a detection method for molecules which are labelled with a fluorescent dye for the detection of dissolved substances or dissolved analytes which behave, for example, like antigen and antibody. This takes the form of a solid-phase sensor with fluorescent dye which permits an energy-transfer process to a molecule which is to be detected and is labelled with a second fluorescent dye.
2. Description of the Related Art
There are various methods for detecting analytes such as hormones, enzymes, other proteins, carbohydrates, nucleic acids, pharmacological active compounds, toxins and others in liquid samples of biological origin. Among the known methods, immunoassays in particular are outstanding as a sensitive detection method for the determination of very small amounts of organic substances. Immunoassay methods are generally based on the ability of a receptor molecule, for example of an antibody, to recognise specifically the structure and molecular organization of a ligand molecule, whether it is defined by non-polar and/or polar interactions, and to bind this molecule very specifically in such a manner.
Immunoassays are carried out by various methods. These include the use of various labelling techniques, usually of a radioactive, enzyme-coupled and fluorescent nature too (Methods in Enzymology, 74 (1981), 28-60) .
Some of these known immunoassay methods entail the use of fluorescent dye molecules F.sub.1 which are able to absorb light of a wavelength .lambda..sub.1 and to emit light of a second, larger wavelength .lambda..sub.2. Under certain conditions, in the presence of another fluorescent dye molecule F.sub.2, excitation of F.sub.1 by light of the wavelength .lambda..sub.1 is followed by a radiationless energy transfer to F.sub.2 which then in turn emits light of a third, even larger wavelength .lambda..sub.3.
This principle of energy transfer has been described in theory by Fo rester and has been the stimulus for a wide variety of possible applications (Annual Reviews in Biochemistry 47 (1978), 819-846). One important property of this energy transfer is its dependence on distance. The efficiency of energy transfer according to Fo rster is described by the critical radius R.sub.o, namely the distance between donor and acceptor at which the intermolecular energy transfer is of equal probability to the total of all other inactivating processes of the donor. This distance is about 50-100 .ANG..
Immunoassays which are based on exploitation of the distance-dependent energy transfer have already been described. Thus, EP 150,905 describes an immunoassay operating in homogeneous solution, in which analyte or antigen has been labelled with a fluorescent dye F.sub.1 and the antibody which binds specifically thereto has been provided with a fluorescent dye F.sub.2. In order to detect the specific binding, and thus as analytical method, use is made of the fact that when light of wavelength .lambda..sub.1 is passed in, emission of the wavelength .lambda..sub.3 can be observed only if analyte and antibody are present in sufficient concentration at a distance which is sufficiently small for energy transfer according to Fo rester. This is the case only when analyte and antibody have entered into specific binding.
In another example, one of the two labelled binding partners is attached to a solid surface, and the correspondingly specifically binding partner is bound from a homogeneous solution. Once again the specific binding is detected as already explained above by an appropriate energy transfer by means of evanescent wave technology (Nature 320 (1986), 179-181).
Both the energy transfer in homogeneous solution, which is mentioned here, and the described solid-phase immunoassay with energy transfer have the disadvantage in principle that the molecules which bind specifically with one another have in each case to be labelled with one of the two necessary fluorescent dyes F.sub.1 and F.sub.2 and, according to Nature 320 (1986), 179-181, allow a maximum F.sub.1 :F.sub.2 ratio of 2:1.
Methods with which the sensitivity, which is limited by the ratio of the two fluorescent dyes F.sub.1 and F.sub.2, of the fluorescent-spectroscopic detection can be improved have already been described. Thus, it is proposed in EP 174,744 that several organic dye molecules be covalently bonded simultaneously to one "light-collecting" protein, that is to say energy transfer of several organic dye molecules to only one acceptor molecule takes place, namely a phycobiliprotein (allophycocyanin) in EP 174,744. This molecular system is then in turn proposed as a "marker" for other biological molecules. The method is limited by the dye:protein coupling ratio.
A further disadvantage of the stated systems derives from the fact that complementary systems have in each case to be specifically labelled and thus versatile use is impossible. Another disadvantage of these systems constructed in heterogeneous phase is the specific evanescent wave technique used. Moreover, the immobilization of the specifically binding molecules to the solid surface via a coupling component/antibody/antigen/antibody system entails very elaborate preparation. Another disadvantage in principle of this solid-phase technology in immunoassays is the reproducible preparation of coatings of the assay matrix with the reactants in the immune reaction. However, besides sensitivity and selectivity for a target substance, an essential quality feature for analytical methods is the reproducibility of the detection method.